Figure 1. (A) View through the thinned skull showing trypanosomes (arrows) and host nuclei labelled with DB75 injected i.v. (B) Graph of relative numbers of trypanosomes as a function of depth below the skull. Means from four areas in one mouse. (C) Movement of trypanosomes in the subarachnoid space tracked over 5 minutes. Some tracks have artifactual gaps.

Trypanosoma brucei, the protozoan that causes African sleeping sickness, can be eliminated from the blood by treatment with available drugs, but in the later stages, after trypanosomes have invaded the brain, current treatments are less successful. To better understand the process of invasion, we have used two-photon microscopy to image trypanosomes in vivo in the meninges and superficial brain through the thinned skull of mice. CD1 mice were injected i.p. with the GVR35 strain of T. brucei, and 3 to 28 days later were injected intravenously with fluorescent markers: dextran-rhodamine to label blood, and the diamidine derivative DB75 which is transported into trypansomes [1]. The mice were then anæsthetised with Hypnorm/Hypnovel (0.05/0.05 mL per gm body weight, i.p.), the anæsthesia being maintained by supplying oxygen carrying gradually increasing amounts of isofluorane (0-2%). A plate with a hole 5 mm in diameter was glued to the skull, and, held by the plate, the skull was thinned over an area about 3 mm in diameter to a thickness of 20-30 μm [2]. Extravascular trypanosomes were observed in clusters in the subarachnoid space centered at a depth below the skull of 22 ± 7 μm (mean ± SD, 6 mice, Figure 1A,B) and they moved mainly within restricted spaces about 30 μm across (Figure 1C). Occasional putative trypanosomes were observed at depths up to 189 μm below the skull; these were closely associated with vessels penetrating into the cortex. Trypanosomes were observed in the subarachnoid space between 7 and 28 days after infection. Over this period the number of trypanosomes per unit area of subarachnoid space did not vary significantly (mean 165 ± 90 mm-2 in 11 mice) although neurological symptoms develop later and progressively. It is not clear how the trypansomes reached the subarachnoid space. Early in brain invasion, trypanosomes are found in the choroid plexus, circumventricular organs and CSF, and there is bulk fluid flow from there to the subarachnoid space [3, 4]. But it has also been found that trypanosomes interact with and cross monolayers of endothelial cells cultured from brain microvessels [5]. Although, in vivo, we often saw intravascular leukocytes adhering to the endothelium of pial venules, we did not observe trypanosomes doing this. However, at one site, in one mouse, we twice observed a brief (<5 sec) release of blood that included a trypanosome.